High sensitivity quantitation of peptides by mass spectrometry

ABSTRACT

The instant invention provides an economical flow-through method for determining amount of target proteins in a sample. An antibody preparation (whether polyclonal or monoclonal, or any equivalent specific binding agent) is used to capture and thus enrich a specific monitor peptide (a specific peptide fragment of a protein to be quantitated in a proteolytic digest of a complex protein sample) and an internal standard peptide (the same chemical structure but including stable isotope labels). Upon elution into a suitable mass spectrometer, the natural (sample derived) and internal standard (isotope labeled) peptides are quantitated, and their measured abundance ratio used to calculate the abundance of the monitor peptide, and its parent protein, in the initial sample.

This application claims priority from U.S. Provisional PatentApplication No. 60/496,037 filed on Aug. 18, 2003; U.S. ProvisionalPatent Application No. 60/449,190 filed on Feb. 20, 2003; U.S.Provisional Patent Application No. 60/420,613 filed on Oct. 23, 2002;and U.S. Provisional Patent Application No. 60/415,499 filed on Oct. 3,2002.

FIELD AND BACKGROUND OF THE INVENTION

This invention relates to quantitative assays for evaluation of proteinsin complex samples such as human plasma. The invention can be used bothfor analysis of samples from a single individual source or, for purposesof evaluating the level of a particular protein in a population, can beused to analyze pooled samples from the target population.

There is a need for quantitative assays for proteins in various complexprotein samples, e.g., in human plasma. Conventionally these assays havebeen implemented as immunoassays, making use of specific antibodiesagainst target proteins as specificity and detection reagents. Newmethods, particularly involving internal standardization withisotopically labeled peptides, allow mass spectrometry (MS) to providesuch quantitative peptide and protein assays (as MS does in themeasurement of low molecular weight drug metabolites currently). Howeverthere remains an issue of the dynamic range and sensitivity of MS assayswhen applied to very complex mixtures, such as those created bydigestion of whole plasma protein to peptides. The present inventionaddresses this problem by providing improvements in sensitivity and byeffectively equalizing the abundances of monitor peptides in a digest ofa sample containing high and low abundance proteins thereby allowingmeasurement of both low and high abundance proteins in a complex sample.

One important advance that can help expand the diagnostically usefulproteome is the use of many protein measurements together as a panel, sothat patterns of change can be associated with disease or treatment,instead of relying on single protein markers interpreted alone. Severalstreams of scientific effort have generated data supporting thisapproach. (See Jellum, Bjornson, Nesbakken, Johansson, and Wold, JChromatogr 217:231-7, 1981.) There were efforts to use the latterapproach to detect disease signatures in then-standard 20-analyte serumchemistry panels, but these met with little success, probably due to theindirect character and small number of the analytes.

The concept and utility of multivariate protein markers has beenestablished for some time. What requires comment is why this approachhas not penetrated significantly into clinical practice.

While proteomics can demonstrate and sometimes measure many proteins,the prior art techniques (e.g., 2D gels) have been difficult to apply toa number of samples large enough to prove a clinical correlation at theresearch level. The alternative approach using existing tests isgenerally too expensive for validating disease correlations of panels.Seventy proteins can all be measured in a single sample of plasma, butthe commercial cost using individual assays is $10,896.30. Thus in theend, the success of multi-analyte diagnostics is as much a matter ofcost as science.

Mass spectrometry (MS) has solved the problem of identifying proteinsresolved by 2D gel and other methods, and appears poised to providegeneral solutions to the analysis of complex protein mixtures as well.In the latter category, two general classes of approach can bedistinguished: first, the “unbiased” discovery of proteins and peptidesachieved via their detection or identification in a sample, and, second,the quantitative measurement of protein or peptides, usually requiringsome type of additional standardization.

The power of mass spectrometry techniques to discover proteins incomplex samples relies, with one notable exception described below, uponthe existence of large protein sequence databases generally derived fromDNA sequencing efforts. Since these databases are becomingcomprehensive, the approach offers, at least in theory, a generalsolution to protein discovery. So far MS efforts have examined threebasic windows into the proteome problem: whole proteins, peptidefragments obtained by digesting proteins in vitro (e.g., with trypsin),and naturally occurring peptides (the low molecular weight proteome, orpeptidome).

Whole proteins can be analyzed by an approach termed SELDI-TOF (forsurface-enhanced laser desorption ionization-time of flight) massspectrometry, a variant of MALDI-TOF (matrix-enhanced laser desorptionionization-time of flight), in which chemical fractionation based onprotein affinity for derivatized MS targets is used to reduce samplecomplexity to a level at which whole-protein MS can resolve a series ofindividual peaks. A significant disadvantage of the approach is that MSanalysis of whole proteins does not directly provide a sequence-basedidentification (there being many proteins with close to a given mass),and hence the protein peaks discovered as markers are notstrictly-speaking identified without significant additional effort. Inparticular, without a discrete identification, it is not generallypossible to demonstrate that a peak is one protein analyte, or totranslate the measurement into a classical immunoassay format. However,as has been clearly demonstrated by the success of some monoclonalantibody-based assays in which the target protein was unidentified, thisdoes not pose a significant limitation to clinical use if the technologyallows the analysis to be repeated in any interested laboratory (aneffort which now appears to be underway).

A more general approach involves digesting proteins (e.g., with trypsin)into peptides that can be further fragmented (MS/MS) in a massspectrometer to generate a sequence-based identification. The approachcan be used with either electrospray (ESI) or MALDI ionization, and istypically applied after one or more dimensions of chromatographicfractionation to reduce the complexity of peptides introduced into theMS at any given instant. Optimized systems of multidimensionalchromatography, ionization, mass spectrometry and data analysis (e.g.,the multidimensional protein identification technology, or “MudPIT”approach of Yates, also referred to as shotgun proteomics) have beenshown to be capable of detecting and identifying ˜1,500 yeast proteinsin one analysis (Washburn, Wolters, and Yates, Nat Biotechnol 19:242-7,2001), while a single dimensional LC separation, combined with theextremely high resolution of a fourier-transform ion cyclotron resonance(FTICR) MS identified more than 1,900 protein products of distinct openreading frames (i.e., predicted proteins) in a bacterium. In humanurine, a sample much more like plasma than the microbial samplesmentioned above, Patterson used a single LC separation ahead ofESI-MS/MS to detect 751 sequences derived from 124 different geneproducts. Very recently, Adkins et al have used two chromatographicseparations with MS to identify a total of 490 different proteins inhuman serum (Adkins and et al, Molec Cell Proteomics 1:947-955 (22002)),thus substantially expanding the proteome. Such methods should have theability to deal with the numerous post-translational modificationscharacteristic of many proteins in plasma, as demonstrated by theability to characterize the very complex post-translationalmodifications occurring in aging human lens.

Naturally-occurring peptides, typically below the kidney filtrationcutoff and hence usually collected from urine or from bloodhemodialysate, provide a complementary picture of many events at thelow-mass end of the plasma proteome. Thousands of liters of humanhemodialysate can be collected from patients with end stage renaldisease undergoing therapeutic dialysis (Schepky, Bensch, Schulz-Knappe,and Forssmann, Biomed Chromatogr 8:90-4, 1994), and even though itcontains only 50 ug/ml of protein/peptide material, it provides alarge-scale source of proteins and peptides below 45 kd. Such materialhas been analyzed by combined chromatography and MS approaches toresolve approximately 5,000 different peptides, including fragments of75 different proteins. Fifty-five percent of the fragments were derivedfrom plasma proteins and 7% of the entries represented peptide hormones,growth factors and cytokines.

The protein discovery methods described above focus on identifyingpeptides and proteins in complex samples, but they generally offer poorquantitative precision and reproducibility. The well-knownidiosyncrasies of peptide ionization arise in large part because thepresence of one peptide can affect the ionization and, thus, signalintensity of another. These have been major impediments to accuratequantitation by mass spectrometry. This problem can be overcome,however, through the use of stable isotope-labeled internal standards.At least four suitable isotopes (²H, ¹³C, ¹⁵N, ¹⁸O) are commerciallyavailable in suitable highly enriched (>98 atom %) forms. In principle,abundance data as accurate as that obtained in MS measurement of drugmetabolites with internal standards (coefficients of variation<1%)should ultimately be obtainable. In the early 1980's ¹⁸O-labeledenkephal ins were prepared and used to measure these peptides in tissuesat ppb levels. In the 1990's GC/MS methods were developed to preciselyquantitate stable isotope-labeled amino acids, and hence proteinturnover, in human muscle and plasma proteins labeled in vivo. Theextreme sensitivity and precision of these methods suggested that stableisotope approaches could be applied in quantitative proteomicsinvestigations, given suitable protein or peptide labeling schemes.

Over the past three years, a variety of such labeling strategies havebeen developed. The most straightforward approach (incorporation oflabel to a high substitution level during biosynthesis), has beensuccessfully applied to microorganisms (Lahm and Langen, Electrophoresis21:2105-14, 2000; Oda, Huang, Cross, Cowburn, and Chait, Proc Natl AcadSci USA 96:6591-6, 1999) and mammalian cells in culture, but is unlikelyto be usable directly in humans for cost and ethical reasons. A relatedapproach (which is applicable to human proteins) is the now-conventionalchemical synthesis of monitor peptides containing heavy isotopes atspecific positions. Post-synthetic methods have also been developed forlabeling of peptides to distinguish those derived from an “internalcontrol” sample from those derived from an experimental sample, with alabled/unlabeled pair subsequently being mixed and analyzed together byMS. These methods include Aebersold's isotope-coded affinity tag (ICAT)approach, as well as deuterated acrylamide and N for labeling peptidesulfhydrals, deuterated acetate to label primary amino groups,n-terminal-specific reagents, permethyl esterification of peptidescarboxyl groups, and addition of twin ¹⁸O labels to the c-terminus oftryptic peptides during cleavage.

Small amounts of proteins such as tissue leakage proteins are importantbecause a serious pathology can be detected in a small volume of tissueby measuring release into plasma of a high-abundance tissue protein.Cardiac myoglobin (Mb) is present in plasma from normal subjects at 1-85ng/mL, but is increased to 200-1,100 ng/mL by a myocardial infarction,and up to 3,000 ng/mL by fibrinolytic therapy to treat the infarct.Cytokines, which in general act locally (at the site of infection orinflammation), are probably not active at their normal plasmaconcentrations (or even at the higher levels pertaining after a majorlocal release) because they are diluted from uL or mL volumes of tissueinto 17 L of interstitial fluid. Hence they are in a sense leakagemarkers as well, though their presence in plasma does not indicate cellbreakage. A commercially useful process for making such measurements isan objective of the instant invention.

The original idea of combining stable isotope labeled peptide internalstandards with an anti-peptide-antibody enrichment step to make aquantitative MS-based assay for a peptide was published in 1989 byJardine et al (Lisek, Bailey, Benson, Yaksh, and Jardine, Rapid CommunMass Spectrom 3:43-6, 1989). The reference discloses use of a singlesynthetic stable isotope labeled peptide (substance P sequence) spikedinto neuronal tissue, followed (after extraction from the tissue) bybinding to an immobilized anti-substance-P-specific antibody, to enrichthe neuropeptide substance P, and finally quantitation by MS. SubstanceP abundance was calculated from the ratio of natural peptide ion currentto the internal labeled standard peptide of the same sequence: i.e.,demonstrating all elements of the single analyte peptidestandard/antibody enrichment process. Jardine et al used a 10-fold molarexcess of the labeled version of substance P to act as both internalstandard and carrier, and measured masses by fast-atom bombardment (FAB)selected-ion monitoring (SIM) MS. As reported, the Jardine approach wasapplied only to endogenous peptides, not in vitro prepared proteinfragments (e.g., a tryptic digest of one or more larger proteins). Theantibody capture was carried out offline, the eluent concentrated andthen applied to a C18 capillary column from which it was eluted into theESI source.

Nelson et al (Intrinsic Bioprobes) have developed similar methods forenriching specific proteins by use of Ab's, and then detecting by MS(with and without added isotope-labeled standards), though they do notmention application to peptides derived by digestion of target proteins.They did assay human beta-2 microglobulin using an antibody to enrichthe protein from plasma, and using equine b2M (from added equine serum)as an internal calibrant (Kiernan, Tubbs, Nedelkov, Niederkofler, andNelson, Biochem Biophys Res Commun 297:401, 2002; Niederkofler, Tubbs,Gruber, Nedelkov, Kiernan, Williams, and Nelson, Anal Chem 73:3294-9,2001a). Nelson (U.S. Pat. No. 5,955,729) has used internal standardpeptides added to samples of affinity purified natural peptides, but inthis case the standard peptides were of different sequence from theanalytes and were not bound on the same antibodies. Both the stableisotope labeled peptides and anti-peptide antibodies are now commonplacereagents, available from multiple commercial sources.

Since 1995 a single peptide has been used as a surrogate for thepresence of a parent protein (from which the peptide was derived byproteolytic digestion) in a complex protein mixture, based on, e.g.,MALDI-PSD (Griffin, MacCoss, Eng, Blevins, Aaronson, and Yates, RapidCommun Mass Spectrom 9:1546-51, 1995) or ion trap (Yates, Eng,McCormack, and Schieltz, Anal Chem 67:1426-36, 1995) MS/MS spectra.

Regnier et al have pursued a “signature peptide” quantitation approach(Chakraborty and Regnier, J Chromatogr A 949:173-84, 2002a; Chakrabortyand Regnier, J Chromatogr A 949:173-84, 2002a; Zhang, Sioma, Wang, andRegnier, Anal Chem 73:5142-9, 2001a), also the subject of a publishedpatent application (Regnier, F. E., X. Zhang, et al. US 2002/0037532),in which protein samples are digested to peptides by an enzyme,differentially labeled with isotopically different versions of a proteinreactive agent, purified by means of a selective enrichment column, andcombined for MS analysis using MALDI or ESI-MS. This method includessome of the features of the present invention, but specifically electsto use post-synthetic labeling of peptides in digests to generate theinternal standards (to allow analysis of unknown peptides), anddescribes the application of antibodies as one of the means forenriching for group-specific characteristics of peptides rather thanunique peptides: “A portion of the protein or peptide amino acidsequence that defines an antigen can also serve as an endogenousaffinity ligand, which is particularly useful if the endogenous aminoacid sequence is common to more than one protein in the originalmixture. In that case, a polyclonal or monoclonal antibody that selectsfor families of polypeptides that contain the endogenous antigenicsequence can be used as the capture moiety” (Regnier, F. E., X. Zhang,et al. US 2002/0037532).

Scrivener, Barry et al (Scrivener, Barry, Platt, Calvert, Masih,Hextall, Soloviev, and Terrett, Proteomics 3:122-128, 2003; Barry et al,U.S. patent application 2002/0055186) have used antibodies fixed on anarray to enrich peptides from a digest for detection by MALDI MS. Thisapproach requires that the antibodies be fixed in a particular spatialform convenient for MALDI MS analysis (generally an array on the surfaceof a planar substrate), and does not include labeled versions of targetpeptides as internal standards for quantitation.

Gygi used stable-isotope-labeled synthetic peptides to quantitate thelevel of phosphorylated vs non-phosphorylated peptides in the digest ofa protein isolated on a 1-D gel (Stemmann, Zou, Gerber, Gygi, andKirschner, Cell 107:715-26, 2001) and has described a method for peptidequantitation (WO03016861) that uses the approach of Jardine with theaddition of greater mass spectrometer resolution (selected reactionmonitoring [SRM] in which the desired peptide is isolated by a firstmass analyzer, the peptide is fragmented in flight, and a specificfragment is detected using a second mass analyzer). Conventionalseparations (eg., reverse phase LC) rather than specific capturereagents (such as antibodies) were to separate peptides prior to MS.

Standards can be made by chemical synthesis. Crowther published asimilar approach in 1994 (Anal Chem 66:2356-61, 1994) to detect peptidedrugs in plasma using deuterated synthetic internal standards. Rose usedsynthetic stable isotope labeled insulin to standardize an MS method forquantitation of insulin (a small protein or large peptide), in which thespiked sample was separated by reverse phase chromatography tofractionate the sample Even larger proteins can now be made by totalchemical synthesis.

Several means for affinity capturing of proteins and peptides usingantibodies are known to the art. Antibody-bound proteins have beeendigested to eliminate non-epitope peptides, followed by elution andidentification of the epitope peptide by MS (Proc Natl Acad Sci USA87:9848-52, 1990). DNA has been used (not an Ab) to bind lactoferrin ininfant urine for analysis by MS (Pediatr Res 29:243-50, 1991).

Protein:protein interactions have previously been mapped by capturingepitope peptides on an antibody, followed by MS (Methods Mol Biol146:439-52, 2000). Methods have been developed for identifying peptideepitopes by allowing an immobilized Ab to subtract the binding (epitope)peptide from a digest prior to MS (J Am Soc Mass Spectrom 11:746-50,2000).

An antibody on magnetic beads has been used to bind a selected protein,which was then digested and the peptides analyzed by MS (J Am Soc MassSpectrom 9:208-15, 1998). Hurst developed a method for solid phaseantibody affinity capture of a protein ligand (TNF-alpha) and subsequentanalysis by MS (Anal Chem 71:4727-33, 1999). Wehland has enrichedpeptides by binding to antibodies and other proteins to identify linearbinding epitopes (Anal Biochem 275:162-70, 1999).

Naylor developed a similar procedure for isolating transferrin prior toMS for the detection of glycosylation variants (Anal Biochem 296:122-9,2001). Clarke and Naylor published (Clarke, Crow, Younkin, and Naylor,Anal Biochem 298:32-9, 2001) a method in which the 40 amino acid amyloidbeta peptide is captured by an antibody to 16 amino acids, eluted andquantitatively detected by MS. The method did not include use of aninternal standard labeled with stable isotopes.

Thibault used a microfluidic device to capture c-myc peptides onantibodies prior to MS, providing detection of spiked peptide to 20ng/ml (Mol Cell Proteomics 1:157-68, 2002).

Recycling immunoaffinity, using immobilized polyclonal antibody columns,has been known since 1975. Using antibodies immobilized onCNBr-activated Sepharose or commercially available POROS supports(Applied Biosystems), polyclonal antibodies have been shown to berecyclable several hundred times without loss of substantial specificbinding capacity.

The instant invention uses several of the cited methods of the prior artin an entirely different combination. In the descriptions that follow,quantitation of proteins, peptides and other biomolecules is addressedin a general sense, and hence the invention disclosed is in no waylimited to the analysis of plasma and other body fluids.

SUMMARY OF THE INVENTION

The instant invention provides an economical flow-through method fordetermining amount of target proteins in a sample. An antibodypreparation (whether polyclonal or monoclonal, or any equivalentspecific binding agent) is used to capture and thus enrich a specificmonitor peptide (a specific peptide fragment of a protein to bequantitated in a proteolytic digest of a complex protein sample) and aninternal standard peptide (the same chemical structure but includingstable isotope labels). Upon elution into a suitable mass spectrometer,the natural (sample derived) and internal standard (isotope labeled)peptides are quantitated, and their measured abundance ratio used tocalculate the abundance of the monitor peptide, and its parent protein,in the initial sample. This is different from the use of a less-specificaffinity method to capture a class of monitor peptides that share aproperty such as glycosylation, inclusion of a cysteine or a lysineresidue, phosphorylation; or use of another form of fractionation thatselects analytes residing in a specific cell fraction, having a similarnative molecular mass (e.g., size exclusion chromatography), charge,etc. Such class-specific fractionation approaches has been exploited byothers including Regnier, where the objective and practice is to reducethe complexity of the mixture presented to the MS somewhat, so as not tooverwhelm its resolution and sensitivity, but not to a single peptideper affinity binding agent (usually antibody). In the present invention,the objective is to select, with a given antibody or other bindingagent, a single peptide derived from a single target protein (or otheranalyte) from the digest of a complex protein sample. The inventionprovides methods for multiplexing peptide measurements, for effectivelyselecting monitor peptides sequences, and for further increasingmeasurement sensitivity.

This disclosure also teaches supports with binding agents which cancollect from a sample target peptides and proteins of varyingconcentration. By selective use of binding agents and amounts of suchagents, it is possible to obtain a large portion of the targetpeptides/proteins which are in small quantities in the sample, whilebinding only a small portion of target peptides/proteins which are inhigh concentration in the sample. This improved method facilitates theefficiency and accuracy of MS reading by narrowing the range ofconcentration of the target proteins or peptides in the elute introducedinto the spectrometer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Each antibody binding surface comprises the lumen of a hole in aplate (here a circular plate), and the lumen's surface area is increasedby fluting. FIG. 1 illustrates the fluting of the antibody-containingholes or tubes to increase surface area.

FIG. 2 shows the arrangement of binding surfaces in holes in disc 8containing teeth 9 to allow its controlled rotation. Shown are sixteenantibody-containing holes 10, and four clear holes 11, aligned aroundaxis 12.

FIG. 3 shows how a set of discs 8 are aligned as stack 17 with clearaligned end caps 16 and 18. Bound antibodies are shown as 19.

FIG. 4. An arrangement for aligning and loading the discs is shown inFIG. 4 where antibody solution 29 in bulb 28 is alternately squeezed andexpanded by device 30 driven under computer control by 31 to push theliquid back and forth through tubes 32,33 and 34, and up through tube 35into bulb 36. The solution pushed through 37 does not build up pressurein bulb 36 because of the presence of hole 38. Antibodies can thus beapplied to a single hole of each of a series of disks, and processrepeated to apply antibodies (typically of different specificities) tothe other holes. The fluted lumen surfaces are chemically modified so asto bind the antibodies. Once the antibodies are applied the holes can bewashed and the antibodies dried in place. The stack of disks is thendisassembled to yield a series of identical antibody-loaded peptidecapture disks.

FIG. 5. In use, as shown in FIG. 5, disc 39 is held as 40 between cleardiscs 41 and 42, containing aligned holes. The arrangement keeps discs41 and 42 stationary while disc 40 can be rotated by engagement ofexternal means with the teeth 9.

FIG. 6. The operation of the system in one analytical cycle is shown inFIG. 6, where disc 63 with teeth 64 contains sixteen antibody-containingholes 65 and four clear holes 70-73.

FIG. 6 b is a circular cross-section through the disks shown in FIG. 6.

FIG. 7 indicates how the entire system in integrated, with the massspectrometer 74 attached to disc analyzer 75, in turn fed discs made andcontrolled by 76, all under the control of computer 77.

FIG. 8 illustrates the abundances of a series of peptide analytes havinga wide range of concentrations in a sample digest (A), addition ofinternal standard peptides in concentrations similar to the expectedanalyte concentrations (B) and the equalization of these concentrationsafter capture and elution from antibody binding media.

FIG. 9 illustrates how groups of peptide analytes occurring at similarconcentrations can be treated together so as to economize on usage ofinternal standards.

FIG. 10 shows how sets of monitor peptides (natural peptides a solidpars, labeled standards as hatched) can be selected from groupings thatdo not overlap in mass, and hence can be separately quantitated.

FIG. 11 illustrates the use of multiple internal standard peptides ofdifferent masses at different concentrations to provide a working curvefor quantitation of a sample peptide analyte.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a flow-through process for identifying andquantitating peptides and/or proteins in a sample. While many of themethods disclosed above are incorporated into the methods of theinvention, the process for such a commercially useful process had notpreviously been disclosed.

The invention is illustrated using the methods to detect proteinanalytes through use of monitor peptides and anti-peptide antibodies,although other sets of reagents can be used to similarly detect otherclasses of analyte molecules. Throughout the disclosure, the terms“analyte”, and “ligand” may be any of a variety of different molecules,or components, pieces, fragments or sections of different molecules thatone desires to measure or quantitiate in a sample. The term “monitorfragment” may mean any piece of an analyte up to and including the wholeanalyte which can be produced by a reproducible fragmentation process(or without a fragmentation if the monitor fragment is the wholeanalyte) and whose abundance or concentration can be used as a surrogatefor the abundance or concentration of the analyte. The term “monitorpeptide” means a peptide chosen as a monitor fragment of a protein orpeptide.

The terms “binding agent” and “receptor” may be any of a large number ofdifferent molecules, biological cells or aggregates, and the terms areused interchangeably. In this context, a binding agent binds to ananalyte being detected in order to enrich it prior to detection, anddoes so in a specific manner, such that only a single analyte is boundand enriched. Proteins, polypeptides, peptides, nucleic acids(oligonucleotides and polynucleotides), antibodies, ligands,polysaccharides, microorganisms, receptors, antibiotics, test compounds(particularly those produced by combinatorial chemistry) may each be abinding agent.

The term “antibody” may be any of the classes of immunoglobulinmolecules of any species, or any molecules derived therefrom, or anyother specific binding agents constructed by variation of a conservedmolecular scaffold so as to specifically bind an analyte or monitorfragment. The term “anti-peptide antibody” may be any type of antibody(in the preceding general sense) that binds a specific peptide, monitorpeptide, or other monitor fragment for the purposes of enrichment from asample or processed sample. In general, any use made of an antibodyherein is understood to be a purpose that could also be served by abinding agent as defined above.

The term “bind” includes any physical attachment or close association,which may be permanent or temporary. Generally, reversible bindingincludes aspects of charge interactions, hydrogen bonding, hydrophobicforces, van der Waals forces. etc., that facilitate physical attachmentbetween the molecule of interest and the analyte being measured. The“binding” interaction may be brief as in the situation where bindingcauses a chemical reaction to occur. Reactions resulting from contactbetween the binding agent and the analyte are also within the definitionof binding for the purposes of the present invention, provided they canbe later reversed to release a monitor fragment.

The terms “internal standard”, “isotope-labeled monitor fragment”, or“isotope-labeled monitor peptide” may be any altered version of therespective monitor fragment or monitor peptide that is 1) recognized asequivalent to the monitor fragment or monitor peptide by the appropriatebinding agent and 2) differs from it in a manner that can bedistinguished by a mass spectrometer, either through direct measurementof molecular mass or through mass measurement of fragments (e.g.,through MS/MS analysis), or by another equivalent means.

The term “specific monitor peptide” refers to a peptide having a uniquesequence in the region of antibody (or other binding agent) contact, andderived from the protein product of a single gene. Peptides havingnon-material modifications of this sequence, such as a single amino acidsubstitution (as may occur in natural genetic polymorphisms),substitutions outside the region of contact or chemical modifications tothe peptide (including glycosylation, phosphorylation, and otherwell-known post-translational modifications) that do not materiallyaffect binding are included in this term. Each antibody preparation (orother binding agent) is meant to enrich a single monitor peptide toserve as the surrogate to a single protein analyte. In order to detectand quantitatively measure protein analytes, the invention makes use ofanti-peptide antibodies (or any other binding entity that can reversiblybind a specific peptide sequence of about 5-20 residues) to capturespecific peptides from a mixture of peptides, such as that arising, forexample, from the specific cleavage of a protein mixture (like humanserum) by a proteolytic enzyme such as trypsin or a chemical reagentsuch as cyanogen bromide. By capturing a specific peptide throughbinding to an antibody (the antibody being typically coupled to a solidsupport), followed by washing of the antibody:peptide complex, andfinally elution of the bound peptide into a small volume (typicallyachieved by an acid solution such as 10% acetic acid), the inventionmakes it possible to enrich specific peptides that may be present at lowconcentrations in the whole digest, and therefore undetectable in simplemass spectrometry (MS) or liquid chromatography-MS (LC/MS) systemsagainst the background of more abundant peptides present in the mixture.This enrichment step is intended to capture peptides of high, medium orlow abundance and present them for MS analysis: it therefore discardsinformation as to the relative abundance of a peptide in the startingmixture in order to boost detection sensitivity. This abundanceinformation, which is of great value in the field of proteomics, can berecovered, however, through the use of isotope dilution methods: theinvention makes use of such methods (preferably using stable isotopes)in combination with specific peptide enrichment, to provide a method forquantitative analysis of peptides, including low-abundance peptides.

The approach is to create a version of the peptide to be measured whichincorporates one or more isotopes of mass different from the predominantnatural isotope, thus forming a labeled peptide variant that ischemically identical (or nearly-identical) to the natural peptidepresent in the mixture, but is nevertheless distinguishable by a massspectrometer because of its altered peptide mass (the isotopic label).The preferred method for creating the labeled peptide is chemicalsynthesis, wherein a peptide identical to the natural one can be made byincorporating amino acid precursors that contain heavy isotopes ofhydrogen, carbon, oxygen or nitrogen to introduce the isotopic label.This isotopic peptide variant is used as an internal standard, added tothe sample peptide mixture at a known concentration before enrichment byantibody capture. The antibody thus captures and enriches both thenatural and the labeled peptide together (having no differentialaffinity for either since they are chemically the same) according totheir relative abundances in the sample. Since the labeled peptide isadded at a known concentration, the ratio between the amounts of thenatural and labeled forms detected by the final MS analysis allows theconcentration of the natural peptide in the sample mixture to becalculated. Thus the invention makes it possible to measure the quantityof a peptide of low abundance in a complex mixture, and since thepeptide is typically produced by quantitative (complete) cleavage of amixture of proteins, the abundance of the parent protein in the mixtureof proteins can be deduced. The invention can be extended to covermultiple peptides measured in parallel, and can be automated throughcomputer control to afford a general system for protein measurement.Creating a new protein-specific assay thus requires only that apeptide-specific antibody and a labeled peptide analog be created. A keyfeature of the invention is that it is directed at establishingquantitative assays for specific proteins selected a priori, rather thanat the problem of comparing all of the unknown components of two or moresamples to one another. It is this focus on specific assays that makesit attractive to generate specific antibodies to each monitor peptide(in principle one antibody binding one peptide for each assay): it iscurrently unattractive to create the thousands to millions of possibleanti-peptide antibodies that would be required to cover the entire rangeof possible human proteins, for example. Previously described methodshave not focused on anti-peptide antibodies for this reason, but usedinstead general affinity concepts that would bind and enrich all of aclass of peptides by recognizing a ligand, label or feature common tothe class: e.g., immobilized metal affinity chromatography (IMAC) toselect phosphopeptides as a group, anti-phosphotyrosine antibodies toselect anti-phosphotyrosine-containing peptides as a group, or lectinsto select glycopeptides as a group. The objective of this invention isto provide means to enrich each peptide sequence specifically with adifferent antibody (or other equivalently selective binding reagent).

A further objective is to deliver a series of different monitor peptides(selected by a corresponding series of specific antibodies) to a massspectrometer at very nearly the same abundance and free of otherextraneous peptides. By equalizing the abundance of a series ofpeptides, the method ensures that all the peptides are within the massspectrometer's dynamic range and that this dynamic range can beoptimally employed in spanning the true dynamic range of the peptideanalytes. If the MS system has a dynamic range of 1000 (a range of 100to 10,000 is typical depending on the type of MS), the method ensuresthat all of the peptides are presented to the MS at a level in themiddle of that range, thus allowing an optimal capacity to detectincreases or decreases in relative abundance of the natural andisotopically labeled forms. If the peptides were presented to the MS atdifferent abundances (e.g., at relative concentrations of 1, 0.001 and1,000), then the MS will have great difficulty in detecting equivalentquantitative differences between natural and isotopically labeled formsof these three peptides. By “flattening” the abundance distribution ofthe peptides, the mass spectrometer's quantitative resolution issubstantially enhanced.

While some skill will be useful in the selection of the optimalpeptide(s) for monitoring each protein, the approach is general andinexpensive relative to the substantial cost of creating thehigh-affinity antibodies and other elements required to perform atypical sandwich-type quantitative immunoassay. It may compete withlow-volume immunoassay technologies as a means of measuring tens tohundreds of specific proteins in mixtures such as human blood serum andplasma.

The preferred embodiment combines 1) existing methods for creation andaffinity purification of antibodies that tightly but reversibly bindshort peptide sequences; 2) existing methods for digestion of complexprotein mixtures to yield short peptides; 3) existing methods forsynthesis of defined peptides containing isotopic labels; 4) existingmethods for efficient recycling affinity chromatography to repeatedlycapture and deliver peptides; and 5) existing methods for MS measurementof ratios of labeled and unlabeled (sample-derived) peptides to yield aquantitative measurement. Herein is described the combined method, usingplasma proteins as an example. In addition to combining the individualsteps in a novel way, we describe novel methods of multiplexing andautomating such assays, and ways of optimizing the choice of monitorpeptide sequences. The application to any other protein or peptidemixture will be obvious to a person skilled in the art. The use ofpeptide-binding agents other than antibodies (e.g., RNA aptamers,peptide aptamers, etc.) will also be obvious to a person skilled in theart. Likewise the generalization of the concept to the quantitativedetection of other biomolecules, such as nucleic acids andoligosaccharides, or to any molecular entity that can be 1) produced inan isotopically labeled form and 2) to which a reversible biding agentcan be created, will be obvious.

Single Analyte Embodiment (1:)

In the simplest embodiment, the following steps are carried out for eachprotein one wishes to measure in plasma in order to generate a specificquantitative assay system. The starting point is a proteinidentification, typically expressed as an accession number in a sequencedatabase such as SwissProt or Genbank. The steps are:

Select Monitor Peptide (Step a)

Using the known sequence of the protein, one selects one or more peptidesegments within it as “monitor peptides”. A good monitor peptide isdefined by a set of criteria designed to select peptides that can bechemically synthesized with high yield, that can be detectedquantitatively in an appropriate mass spectrometer, and that elicitantibodies when used as antigens, although any peptide resulting fromcleavage with the desired enzyme is a possible choice. One useful set ofcriteria is the following:

1: The peptide has a sequence that results from cleavage of the proteinwith a desired proteolytic enzyme (e.g., trypsin). All the candidatetryptic peptides can be easily computed from the protein sequence byapplication of generally available software.

2: The peptide should be hydrophilic overall, and soluble inconventional solvents used in enzymatic digestion and affinitychromatography. Hydrophilic peptides can be selected based on computedscores obtained for each peptide from generally available softwareprograms. In general the hydrophilic peptides are those that containmore polar amino acids (his, lys, arg, glu, asp) and fewer hydrophobicamino acids (trp, phe, val, leu, ile).3: The peptide should preferably contain no cys, as a c-terminal cys maybe added for convenience in conjugation of the immunogen, and thepresence of two cys in a peptide can lead to undesirable dimerizationand cross-linking.4: the peptide should ionize well by either electrospray (ESI) ormatrix-assisted laser desorption (MALDI) ionization. This characteristiccan be estimate by software programs or determined experimentally by MSanalysis of a digest of the protein in question to see which peptidesare detected at highest relative abundance.5: The peptide should be immunogenic in the species in which theantibody will be raised. Immunogenicity is generally better for peptidesthat are hydrophilic (compatible with (2) above); that include a bendpredicted by secondary structure prediction software; that include noglycosylation sites; and that are 10-20 amino acids in length.6: The peptide should not include within it the sites of any commonsequence polymorphisms (i.e., genetic variants) in the target protein(as this could affect the estimation of the respective protein'sabundance if the variant peptide does not appear at the expected mass).7: the peptide should not share appreciable homology with any otherprotein of the target organism (as determined for example by the BLASTsequence comparison program). This characteristic should tend to reduceany interference in the antibody capture step from peptides originatingin proteins other than the target.

All possible peptides derived from the target protein can easily beevaluated according to these criteria and one or more peptides selectedthat best balance the requirements of the method. Specifically it isstraightforward to create a database of all the peptides and theirderived properties for a finite set of analytes such as the knownproteins in plasma, and to use this database as a basis for selection ofmonitor peptides. Beginning with the known amino acid sequences ofprotein analytes, efficient algorithms can construct all the possibletryptic peptides that will be created by trypsin digestion of theprotein. These tryptic peptide sequences can be stored as records in adatabase, and similar records generated for other possible cleavageenzymes and reagents. Additional algorithms can be employed to computevarious physical and biological properties of each peptide, includinglength, mass, net charge at neutral pH, propensity to adopt secondarystructure, hydrophilicity, etc. These derived data can be tabulated foreach peptide, and additional aggregate calculations performed to developprioritizing scores associated with likelihood of success as a monitorpeptide. These priority scores can be sorted to select preferredcandidate monitor peptides for each protein.

It is also possible to add experimental data to the prioritization. Itis possible, for example, to generate by synthesis all the individualtryptic peptide sequences derived from a protein, and to immobilizethese peptides in an array on a membrane. Such as array can then beprobed with an antibody to the whole protein, or with a mixture ofantibodies raised against a mixture of proteins including the targetprotein, and the binding of antibody to the various peptides revealedand quantitated by secondary staining with a second antibody labeled soas to be detectable by luminescence, fluorescence, or colorimetricstaining (the PEPSCAN approach {Carter Methods Mol Biol 36: 207-223(1994). Those peptides situated where antibody binding is detected arethus shown to be capable of eliciting and antibody response. The majorlimitation to thus using PEPSCAN is the requirement for an existingantibody to the protein of choice, or to a mixture containing it. Suchan antibody may be available when the protein is one that has beenstudied before, or it may be generated in conjunction with an attempt toselect the optimal monitor peptide.

Creating Isotope Monitor Peptides (Step b)

An isotopically labeled version of the selected peptide(s) is then madein which the chemical structure is maintained, but one or more atoms aresubstituted with an isotope such that an MS can distinguish the labeledpeptide from the normal peptide (containing the natural abundance ofeach elements' isotopes). For example, nitrogen-15 could be introducedinstead of the natural nitrogen-14 at one or more positions in thesynthesized peptide. The synthesized peptide will be heavier by a numberof atomic mass units equal to the number of substituted nitrogens. Thepeptide is carefully made so that the number of added mass units isknown and well-determined (i.e., all of the material produced as onestandard has the same mass insofar as possible—achieved by using highlyenriched isotopic variants of the amino acids, for example). In thepreferred embodiment, nitrogen-15 labeled amino acid precursorssubstituted at >98% are used at one or more positions in the peptidesynthesis process to introduce between 4 and 10 additional mass unitscompared to the natural peptide. Such nitrogen-15 labeled amino acidprecursors (or their carbon-13 labeled equivalents) are commerciallyavailable as FMOC derivatives suitable for use directly in conventionalcommercial peptide synthesis machines. The resulting labeled monitorpeptides are purified using conventional LC methods (typically to >90%purity) and characterized by MS to ensure the correct sequence and mass.

Creating Anti-Peptide Antibodies (Step c)

To immunize an animal for production of anti-peptide antibodies, thesame peptide (labeled or not, if this is, as expected, more economical)is coupled to a carrier protein (e.g., keyhole limpet hemocyanine (KLH);not homologous to a human protein) and used to immunize an animal (suchas a rabbit, chicken, goat or sheep) by one of the known protocols thatefficiently generate anti-peptide antibodies. For convenience, thepeptide used for immunization and antibody purification preferablycontains additional c-terminal residues added to the monitor peptidesequence (here abbreviated MONITOR), e.g.:nterm-MONITOR-lys-gly-ser-gly-cys-cterm. The resulting extended monitorpeptide can be conveniently coupled to carrier KLH that has beenpreviously reacted with a heterobifunctional reagent such that multipleSH-reactive groups are attached to the carrier. In classicalimmunization with the peptide (now as a hapten on the carrier protein),a polyclonal antiserum will be produced containing antibodies directedto the peptide, to the carrier, and to other non-specific epitopes.Alternatively, there are many methods known in the art for coupling apeptide, with or without any extensions or modifications, to a carrierfor antibody production, and any of these may be used. Likewise thereare known methods for producing anti-peptide antibodies by means otherthan immunizing an animal with the peptide on a carrier. Any of thealternatives can be used provided that a suitable specific reversiblebinding agent for the monitor peptide is produced.

Specific anti-peptide antibodies are then prepared from this antiserumby affinity purification on a column containing tightly-bound peptide.Such a column can be easily prepared by reacting an aliquot of theextended monitor peptide with a thiol-reactive solid support such ascommercially available thiopropyl Sepharose. Crude antiserum can beapplied to this column, which is then washed and finally exposed to 10%acetic acid (or other elution buffer of low pH, high pH, or highchaotrope concentration) to specifically elute antipeptide antibodies.These antibodies are neutralized or separated from the elution buffer(to prevent denaturation), and the column is recycled to physiologicalconditions for application of more antiserum if needed.

The peptide-specific antibody is finally immobilized on a column, beador other surface for use as a peptide-specific affinity capture reagent.In the preferred embodiment, the anti-peptide antibody is immobilized oncommercially available protein A-derivatized POROS chromatography media(Applied Biosystems) and covalently fixed on this support by covalentcrosslinking with dimethyl pimelimidate according to the manufacturer'sinstructions. The resulting solid phase media can bind the monitorpeptide specifically from a peptide mixture (e.g., a tryptic digest ofserum or plasma) and, following a wash step, release the monitor peptideunder mild elution conditions (e.g., 10% acetic acid). Restoring thecolumn to neutral pH then regenerates the column for use again onanother sample, a process that is well known in the art to be repeatablehundreds of times.

The preferred affinity of the anti-peptide antibodies is in the range of100 to 100,000,000. A higher affinity is required to enrich lowerabundance peptides, i.e., to capture peptides at low concentration.

Digestion of Sample to Peptides (Step d)

A sample of plasma, in which one wishes to measure the selected protein,is digested essentially to completion with the appropriate protease (inthis case trypsin) to yield peptides (including the monitor peptideselected in step 1). For a monitor peptide whose sequence appears oncein the target protein sequence, this digestion should generate the samenumber of monitor peptide molecules as there were target proteinmolecules in the stating sample. The digestion is carried out by firstdenaturing the protein sample (e.g., with urea or guanidine HCl),reducing the disulfide bonds in the proteins (e.g., with dithiothreitolor mercaptoethanol), alkylating the cysteines (e.g., by addition ofiodoacetamide), quenching excess iodoacetamide by addition of moredithiothreitol or mercaptoethanol, and finally (after removal ordilution of the denaturant) addition of the selected proteolytic enzyme(e.g. trypsin), followed by incubation to allow digestion. Followingincubation, the action of trypsin is terminated, either by addition of achemical inhibitor (e.g., DFP or PMSF) or by denaturation (through heator addition of denaturants, or both) or removal (if the trypsin is on asolid support) of the trypsin. The destruction of the trypsin activityis important in order to avoid damage to antibodies later by residualproteolytic activity in the sample.

Adding Isotopically-Labeled Monitor Peptide Internal Standards (Step e)

A measured aliquot of isotopically-labeled synthetic monitor peptide isthen added to a measured aliquot of the digested sample peptide mixturein an amount close to or greater than (if the standard serves as carrierfor a low abundance peptide) the expected abundance of the same“natural” peptide in the sample aliquot. Following this addition themonitor peptide will be present in the sample in two forms (natural andisotopically-labeled). The concentration of the isotopically-labeledversion is accurately known based on the amount added and the knownaliquot volumes.

Enrichment of the Monitor Peptide by Antibody Capture and Elution (Stepf)

The peptide mixture (digest with added isotopically-labeled monitorpeptides) is exposed to the peptide-specific affinity capture reagent,which preferentially binds the monitor peptide but does not distinguishbetween labeled and unlabeled forms (since isotopic substitutions arenot expected to affect antibody binding affinity). Following a wash step(e.g., phosphate-buffered saline) the bound peptides are then eluted(e.g., with 10% acetic acid, or with a mixture of water andacetonitrile), for MS analysis. The affinity support can, if desired, berecycled in preparation for another sample. In the high-throughput assayapplications envisioned, it will be advantageous to recycle theimmobilized antibody binding hundreds, if not thousands, of times.Current evidence indicates that rabbit polyclonal antibodies can berecycled at least 200 times when antigens are eluted with 5% or 10%acetic acid and total exposure to acid is kept short (e.g., less than 1minute before regeneration with neutral pH buffer). In a capillarycolumn format, where the immobilized antibody bed can be submicroliterin size, the duration of acid exposure could be further decreased,possibly extending the life of the immobilized antibody adsorbent evenfurther.

The efficiency of peptide capture is governed by the affinity constantof the antibody for the peptide and by the concentrations of bothpeptide and antibody. We are concerned particularly with the fraction ofpeptide that is combined with antibody. The relevant general equationsare:Ab+Pep=AbPep,K _(a) =[AbPep]/([Ab]×[Pep])K _(a) ×[Ab]=[AbPep]/[Pep]

[Ab], [Pep] and [AbPep] are the concentrations of Ab, Pep, AbPep,respectively; and K_(a) is the affinity constant governing the bindingreaction under the solution conditions given. In the present embodimentwe are concerned particularly with low abundance peptides (since highabundance peptides will be relatively easy to capture), and thus wearrange that the antibody is present at the maximum concentrationobtainable on the solid support ([Ab]=approximately 10⁻⁵ M for IgG boundto Protein A derivatized POROS resin). Since at this concentration 100fmol of Ab occupies only 1 nL of POROS, and since the columns actuallyused, though very small by conventional standards, are much larger thanthis, the antibody will be present in substantial excess over thepeptide, and we can assume any attainable level of [AbPep] will notsignificantly decrease [Ab], which can be assumed to be constant (10⁻⁵M). Typical antipeptide antibodies have affinity constants in the range10⁶-10⁸. Hence the ratio of amount of antibody-bound peptide to freepeptide ([AbPep]/[Pep]) is given by K_(a)×[Ab]=10⁶-10⁸×10⁻⁵=10 to 10³. Amajority (90%) of peptide should thus be bound to antibody even with arelatively low affinity (K_(a)=10⁶) antibody, while a high afffintyantibody gives 99.9% antibody binding. This ratio is independent of theantibody's concentration: the antibody captures whatever peptide isavailable, and the sensitivity is determined primarily by the detector'ssensitivity.

The above calculations apply to equilibrium binding. The affinityconstant K_(a) is the quotient of the “on-rate” K_(on) and the“off-rate” K_(off) (K_(a)=K_(on)/K_(off)). K_(n) is similar for allpeptide-antibody binding reactions since it is determined mainly bydiffusion (the molecules bumping into one another). The typical value ofK_(n) is 10⁵ to 10⁶ M⁻¹ sec⁻¹, and here we assume the more conservativevalue (10⁶). From this and the typical values of K_(a) used above(10⁶-10⁸) we can calculate the range of values for K_(off): 1 to 0.01per second, or in other works 1 to 100 seconds. If the peptide canrebind to another binding site after it comes off, then it will staybound for another similar period. Thus it is likely that the peptidewill stay on the column during loading and washing provided these arerelatively fast and provided that there are excess binding sites(antibodies) for re-binding. Elution can be very fast because elutionconditions (e.g., low pH) alter the peptide:antibody interaction anddrastically increase the off rate. For this reason an antigen bound toan immobilized antibody column is observed to elute in a sharp frontalzone even during rapid recycling affinity chromatography. Such frontalbehavior allows elution of captured peptide in a very small volume,particularly if the ratio of column length to diameter is large. Thislater requirement is met by a capillary immobilized antibody columnhaving, e.g., diameter 100 microns and a length of 3 mm(length/diameter=30, volume=˜25 nL). In such a column a peptide zone canbe eluted in a 1 mm zone, having a volume of 8 nl. If the digest of a 10uL plasma sample can be loaded on the column, the monitor peptidecaptured with high efficiency as described above, and then eluted in 8nL, than the method of the invention achieves a concentration increaseof 1,000-fold and simultaneously removes a large amount of potentiallyinterfering peptide material.

The enrichment step is an important step of the method because it allowsenrichment and concentration of low abundance peptides, derived from lowabundance proteins in the sample. Ideally, this enrichment processdelivers only the monitor peptide to the MS, and makes its detection amatter of absolute MS sensitivity, rather than a matter of detecting themonitor peptide against a background of many other, potentially muchhigher abundance peptides present in the whole sample digest. Thisapproach effectively extends the detection sensitivity and dynamic rangeof the MS detector in the presence of other high abundance proteins andpeptides in the sample and its digest.

Analysis of the Captured Monitor Peptides by MS (Set g)

The monitor peptide (including natural and isotopically-labeledversions) enriched in the preceding step is delivered into the inlet ofa mass spectrometer, preferably by electrospray ionization. In apreferred embodiment, the peptide is introduced directly into the massspectrometer in the elution buffer (e.g., 10% acetic acid).Alternatively the monitor peptide is applied to a reverse phase (e.g.,C18) column and eluted by a gradient (e.g., ofacetonitrile/trifluoroacetic acid in water) into an electrospray sourceof the mass spectrometer (i.e., LC/MS). The mass spectrometer can be anion trap, a triple quadrupole, an ESI-TOF, a Q-TOF type instrument, orany other instrument of suitable mass resolution (>1,000) andsensitivity.

The MS measures the ion current (number of ions) for both versions ofthe monitor peptide (natural and labeled) as a function of time. The ioncurrent may be integrated over time (ideally for as long as the monitorpeptide appears in the mass spectrum) for each mass species, and theintegrated amounts of natural and isotope-labeled forms are computed.

Computation of Abundance of Each Monitor Peptide in the Sample (Step h)

A ratio is computed between the amounts of the labeled and unlabeled(natural) monitor peptides. Since the amount of labeled peptide added isknown, the amount of the natural monitor peptide derived from the sampledigest can then be calculated by multiplying the known concentration oflabeled monitor peptide by this measured ratio. By assuming that theamount of the monitor peptide in the digest is the same as (or closelyrelated to) the amount of the parent protein from which it is derived, ameasure of the protein amount in the sample can be obtained.

In order to detect and compensate for variation in the completeness ofthe sample digest process, a series of digest monitor peptides can beselected that indicate the progress of the digestion process. Digestioncompleteness can vary due to differences between sample digests in theratio of proteolytic enzyme to plasma protein, to differences in timeand temperature of digestion, to differences in the samples' endogenousprotease inhibitor content, or to differences in the levels oractivation of endogenous proteases. Specifically, one can carry out atime-course experiment in which a plasma sample is digested by trypsin(after reduction and alkylation of the plasma proteins), and the amountsof each of a series of peptides released can be measured as a functionof time. Some peptides are released early in the course of the digest,probably because they are located at the surface of the target proteinand because the cleavage sites at the peptides' ends are exposed to theprotease, and reach their maximum final concentration in the digestearly. Other peptides are release later during the course of thedigestion, probably because they form part of the core of a targetprotein or because the cleavage sites defining the peptides' ends arenot exposed at the surface of the intact target protein, and thus thesepeptides appear later during the course of digestion and reach theirmaximum final concentration later. It also occurs that some trypticpeptides are released from target proteins that are then further cleavedin solution, leading to an increase in a peptide's concentration in thedigest followed by a decrease later as the peotide is further cleaved toother shorter peptides. By measuring the time vs concentration profilesof a series of specific peptides during such a time course, one canselect digest monitor peptides that together give an accurate measure ofthe status of the plasma digestion process. The utility of a panel ofsuch peptides is increased if they are products of one or a fewproteins, so that abundance ratios between the peptides are reflectiveof the digest progress and not of the differences in concentrationbetween the parent proteins. By measuring the selected digest monitorpeptides in subsequent individual sample digests, one can compute wherein the process of digestion each sample was arrested, effectivelygenerating a standardized scale of digestion progress for the sampletype involved. This information, in combination with knowledge of thetime course for release of each monitor peptide to be used in sampleanalysis (relative to release of the digest monitor peptide in areference digest), will allow corrections to be applied to monitorpeptide abundances when specific samples are not digested to exactly thesample extent.

The specificity and sensitivity of the approach can be further enhancedby use of a multistage peptide capture methodology. In one embodiment ofa multistage approach two different sequential capture steps can be usedbased on a first capture on an antibody raised to the N-terminal portionof the peptide and, after elution from this first antibody andneutralization, capture on a second antibody (A2) raised against theC-terminal portion of the peptide. Such N-term or C-term antibodies canbe made because the immunogen typically used to make antipeptideantibodies consists of the peptide coupled to a large carrier protein(e.g., keyhole limpet hemocyanine or albumin) through a cysteine residueappended to the sequence of the desired monitor peptide (often spacedapart from the monitor peptide sequence by some spacer residues). If thecysteine is included at the N-terminus of the immunizing peptide,causing the N-term to be attached to the carrier surface, the C-terminusof the monitor peptide sequence will be exposed for recognition andantibody generation. Conversely, if cysteine is included at theC-terminus of the immunizing peptide, causing the C-term to be attachedto the carrier surface, the N-terminus of the monitor peptide sequencewill be exposed for recognition and antibody generation. Sinceantibodies often recognize a stretch of 3 to 6 amino acids, and sincethe monitor peptides are often 8 to 15 amino acids long, there is a highprobability that the epitopes recognized by the N-term and C-termantibodies will be substantially different, thus offering separate butspecific recognition of the monitor peptide. Any impurities (otherpeptides) that bind to the first (e.g., N-term) antibody through somesimilarity to the N-terminal portion of the monitor peptide sequence arevery unlikely to bind also to the C-term antibody. Using two separateenrichment processes will in general give a purification equal to theproduct of the two separate enrichment steps: if the N-term antibodybinds 1 part in 10⁴ of the digest peptides (as impurities, i.e., apartfrom the desired monitor peptide) and the C-term antibody also binds 1part in 10⁴ of the digest peptides (as impurities, i.e., apart from thedesired monitor peptide), then the sequential combination of theseantibodies as two separate enrichment steps is likely to bind 1 part in10⁸ of the non-monitor peptides. If both the N-term and C-termantibodies bind a high proportion of the monitor peptide (e.g., 90% ateach step), then the final result of the two-stage capture would be 81%of the monitor peptide recovered, with only 0.000001% (1 part in 10⁸) ofother peptides bound.

Other multistage enrichment processes can also be beneficial. A firstantibody can be raised either against a surface peptide or else againstthe whole protein) can be used to capture the native protein from plasmaafter which the protein can be digested to peptides, and a monitorpeptide captured by a second anti-peptide antibody.

Alternatively a sequential digestion approach can be used in combinationwith two anti-peptide antibodies. Here the plasma sample is digestedwith a first protease yielding a first version of a monitor peptidesequence that is bound by a first anti-peptide antibody. Followingelution of this peptide, it is cleaved by a second protease, yieldingtwo (or possibly more) new peptides, each of which has at least one newterminus (the prior C-term segment of the first monitor peptide has anew N-terminus, and the prior N-term segment has a new C-terminus). Asecond anti-peptide antibody is used to capture one of these newlyexposed terminal sequences (a terminus that was not exposed prior to thesecond digestion step) for MS detection. One implementation of thisapproach would, as an example, involve selection of a monitor peptidesequence bounded by lysines and/or N- or C-termini, and within whichthere was one arginine residue. Using lys-C (which cleavespreferentially at lysine residues, but not arginine) as the firstprotease, a first anti-peptide antibody would be made to recognize theC-terminal lysine portion of the sequence (e.g., immunizing peptidelinked to carrier via N-term cysteines). A second digestion, carried outwith trypsin (which cleaves at both lysine and arginine) would cleave atthe internal arginine in this peptide, creating two fragments, one ofwhich has a new C-terminal sequence (ending in arginine) and which wouldbe recognized by the second anti-peptide antibody. This approachactually makes use of three specificity steps (first antibody, secondprotease, second antibody) to further increase the overall specificityof the final detection process. In using a two-protease multistagesystem, the opportunity exists to capture and detect two (or more, ifthe second protease cuts more than once) “daughter” peptides separatelyas mutually confirmatory assays. Any combination of proteases withdifferent specificities could be used.

A particularly interesting instance of the two-protease multistagesystem is one in which the action of the first protease occurs in vivo,generating a cleavage in a fraction of the native protein molecules inthe sample. The action of such a protease could be an indication of adisease process or of a beneficial response to therapy, for example. Onedesired measurement would be the fraction of the molecules that werecleaved. This can be achieved by digesting the plasma sample with aprotease (the in vitro protease) that does not cleave at the site of thein vivo protease, generating fragments of the native protein (peptides),one of which contains the in vivo cleavage site. An anti-peptideantibody directed to the N-terminus (or alternatively the C-terminus) ofthe monitor peptide can capture both the entire monitor peptide and theshorter version arising from in vivo cleavage. The ratio in abundancebetween the long and short forms of the monitor peptide (obtainedpreferably by quantitating each against an identical stable isotopelabeled internal standard peptide) gives the ratio of the uncleaved toin vivo cleaved forms of the parent protein.

All of the multistage processes described above can make use ofisotopically labeled peptides as internal standards, in the same waythey are used for quantitation of peptides by the first single-analyteembodiment.

Automation of the Basic Implementation (2:)

Steps d, e, f and g of the basic implementation are preferably combinedinto an automated process, using a computer-controlled fluid handlingsystem for steps d, e, and f, and a computer-controlled massspectrometer for step g. In this approach the computerized fluidhandling system carries out the reduction and alkylation of the sample,addition of trypsin, incubation, quenching of the trypsin activity, andaddition of the labeled peptide standard(s).

In one version, the computerized fluid handling system then applies theprepared digest sample to an antibody (preferably on a solid support)specific for a monitor peptide, removes the digest, washes the antibodyon its support, and finally elutes the captured peptides directly intothe mass spectrometer for step g. The elution can be carried out in avery small volume (e.g., 10 ul) and thus the entire eluted sample can beinstilled into the MS for maximum sensitivity.

Alternatively step f can be carried out offline, generating a series ofenriched peptide samples that can be introduced into a mass spectrometerlater for measurement. This approach may be particularly appropriatewhen a MALDI MS is to be used for detection and quantitation of thepeptides, since a MALDI target plate holding hundreds of samples can beprepared offline and introduced at a later time into the MS.

The entire process of steps a-h can be carried out as a unifiedanalytical process for the quantitation of proteins in a sample.

Parallelized Embodiments for Multiple Analyte Measurement

In a first parallelized embodiment, multiple proteins can be measuredusing individual antibodies to select individual monitor peptides one ata time, in an apparatus that allows successive antibodies to be elutedat intervals into the MS (each monitored peptide is measured insuccession as its antibody is put in position for elution). In thisversion, instead of a chromatography separation to separate a mixturecontaining a series of monitor peptides (in each case together withtheir added isotopically labeled versions), one uses a fluidic ormechanical means to place each antibody, on its solid support, into theelution path (typically a liquid stream of 10% acetic acid eluentdirected into the MS). This version of the basic embodiment can beimplemented using a multiplicity of small antibody columns arranged likethe chambers of a revolver, as shown in FIGS. 1-7.

In this embodiment, two of the most time consuming and expensiveprocesses are the preparation of the digested microsamples, and thepreparation and use of the immobilized antibody surfaces that bind thepeptides to be analyzed. Therefore one would like to prepare only one ora very few digests of a sample to be analyzed, and apply it to as manyimmuno-absorptive supports or surfaces as is efficient and necessary tomeasure the desired number of monitor peptides (target proteins). Sincebinding is diffusion related, an objective is to spread the peptidedigest samples over a relatively large surface, and to both apply andremove it efficiently. Two limitations are evident at the outset. Thefirst is the capacity and polyspecificity of the absorptive surface, andthe second is the limitation of the mass spectrometer to simultaneouslyquantitate a large number of different analytes when increasing thenumber to be detected results in their greater dilution. Thus, if amaximally polyvalent absorptive surface is used, then its capacity forany one analyte is very small, and only a very small amount of eachbinding agent will be present. And as the number of analytes rises to alarge number, the MS may not be able to resolve all the substancespresent. In the present embodiment incorporating automation, theobjective is to arrange for digested samples to pass over a series ofdifferent immunoabsorptive supports, each containing one or a number ofdifferent specific antibodies, in a closed system in such a manner thatall the supports are exposed to the samples, and are then washed free ofexcess sample. After this step, the discrete supports are separated, andeach support eluted separately and efficiently into an electrospray MSsystem. The immunoabsorptive supports must be designed in such a mannerthat the attachment of antibodies to large sets of them (i.e., thecritical step of their manufacture) may be done in parallel, and with ahigh degree of reproducibility.

In the initial design sixteen different immunoabsorptive supportscomprising differing surfaces are used, and it is anticipated that amixture of ten different antibodies will be attached to each, giving atotal of 160 different proteins to be analyzed for in each assay set.Each antibody binding surface comprises the lumen of a hole in a plate(here a circular plate), and the lumen's surface area is increased byfluting. FIG. 1 illustrates the fluting of the antibody-containing holesor tubes to increase surface area.

FIG. 2 shows the arrangement of binding surfaces in holes in disk 8containing teeth 9 to allow its controlled rotation. Shown are sixteenantibody-containing holes 10, and four clear holes 11, aligned aroundaxis 12.

FIG. 3 shows how a set of disks 8 are aligned as stack 17 with clearaligned end caps 16 and 18. Bound antibodies are shown as 19.

An arrangement for aligning and loading disks is shown in FIG. 4 whereantibody solution 29 in bulb 28 is alternately squeezed and expanded bydevice 30 driven under computer control by 31 to push the liquid backand forth through the tubes 32, 33 and 34, and up through tube 35 intobulb 36. The solution pushed through 37 does not build up pressure inbulb 36 because of the presence of hole 38. Antibodies can thus beapplied to a single hole of each of a series of disks, and the processrepeated to apply antibodies (typically of different specificities) toother holes. The fluted lumen surfaces are chemically modified so as tobind antibodies. Once the antibodies are applied the holes can be washedand the antibodies dried in place. The stack of disks is thendisassembled to yield a series of identical antibody-loaded peptidecapture disks.

In use, as shown in FIG. 5, disk 39 is held as 40 between clear disks 41and 42, containing aligned holes. The arrangement keeps disks 41 and 42stationary while disk 40 can be rotated by engagement of external meanswith teeth 9.

The operation of the system in one analytical cycle is shown in FIG. 6,where disk 63 with teeth 64 contains sixteen antibody-containing holes65 and four clear holes 70-73. The antibody-containing holes arealternatively connected by slots in the upper and lower clear disks,with the lower slots indicated by dashed line 66 and the upperconnections by non-dashed lines 67, making a continuous path connectinginlet 68, through all the antibody-containing holes 65 via theserpentine connections of over (67) and under (66) connecting slots, andexiting through 69. This allows a sample digest to be pumped back andforth through all the holes, then expelled, and the hole-set washed.These elements are shown in FIG. 6 b as a circular cross-section throughthe disks. In next operation, the rotatable disk 63 is moved such thatthe antibody loaded tube 69 is aligned with tube 70 of plates 41 and 42,and a wash solution is run through. Indexing one step forward moves tube69 to position 71 where an eluting solution, such as 10% acetic acid, isused to detach the bound peptides and transport them to a massspectrometer, or intermediate capture point, such as reverse phasecolumn. A next indexing brings tube 69 into register with position 72where a buffer is run through to recycle the antibody and render itstable after exposure to the eluting solution. Each differentantibody-containing tube (or hole) is thus indexed through the stations,resulting in a sequence of 16 successive elutions into the MS.

FIG. 7 indicates how the entire system is integrated, with the massspectrometer 74 attached to disk analyzer 75, in turn fed samples byautosampler 76, all under control of computer 77.

The antibody-containing holes can be formed so as to carry antibodies(or other binding agents) on their inner surfaces (as shown) or they canbe filled with a porous monolithic material to which the antibodies arebound, yielding a larger surface area and thus a higher localconcentration of antibody molecules. Such monolithic supports can beformed by polymerization in situ, by sintering pre-made particles, byinsertion of a preformed porous rod into each hole with a friction fit,or by other methods. In the case of antibodies bound to inner surfaces,these surfaces can be cylindrical (as in a normal tube) or they can bereticulated in various ways to increase inner surface area.

In clinical chemistry where a number of different analytical componentssuch as the disks must be used, the most serious problem is that ofbeing sure that the component in place is the correct one. Note thatbuilt into this system is an inherent capability for quality control andpositive identification of binding sites. This is achieved as anautomatic feature of the system because a mixture of standards is addedto each peptide digest sample, only some of which bind to each antibodyloaded binding surface. The analysis of the peptides eluted from eachsurface (hole) by the mass spectrometer therefore provides positiveidentification of the antibodies on that surface, and their operationalcondition. This aspect assures that the disk contains the correctantibody specificities. An added optional feature is the loading of theantibodies in each hole with their corresponding peptide duringmanufacture: in this case each hole in the disk could be eluted into theMS before any samples are loaded, and the antibody specificitiesconfirmed by MS identification of these eluted peptides.

Alternatively the multiple-antibody-separate-elution approach can beimplemented in other ways, for example employing electromagnetic meansto move antibody-coated diamagnetic particles through the requiredpositions (sample, wash, elution into MS). Arrays of immobilizedanti-peptide antibodies, arranged on a flat surface so as to capturepeptides from an overlying fluid volume; arranged on pins so as tocapture peptides from a vessel into which they are dipped; or arrangedin separate microvessels of a microfluidics device so as to beconnectable into multiple fluid flowpaths can also be used. Amultiplicity of mechanical and electromagnet solutions will be apparentto the problem of exposing multiple, separately immobilized antibodiesto a sample, washing them and then eluting them individually by exposureto a stream of liquid that then moves into an MS.

In a second parallelized embodiment, the approach is applied to a seriesof monitor peptides (A-L) of different masses, for measurement of aseries of proteins (a-l), at once (FIG. 8A). In this case a cocktail oflabeled peptides in predetermined amounts (based on expected relativeabundances of the respective proteins in the sample) would be added tothe sample digest (FIG. 8B: solid bars represent the natural peptide,and dashed bars represent the added stable isotope labeled monitorpeptides). A series of capture antibodies would be used to capture justthese monitor peptides (natural and labeled forms). These antibodies (onappropriate solid phase media) would preferably be combined in relativeamounts so as to capture approximately equal amounts of each monitorpeptide, irrespective of the amount of these peptides in the digest,thus resulting in an approximately equimolar mixture of monitor peptidesupon elution into the MS (FIG. 8C). If the antibodies are of highaffinity, then this objective can be achieved by preparing a column, orother affinity support on which approximately equal amounts of eachantibody are fixed, and passing over this support enough sample digestso that all the antibodies can bind to saturation. If one or more of theantibodies has a lower affinity, then more of that antibody may berequired in order to achieve approximately equal stoichiometry ofcaptured and released peptides. Most of the mass of abundant monitorpeptides will therefore not be bound (exceeding the amount of captureantibody on the support), but the low abundance peptides may only justsaturate the respective capture antibody with none appearing in theflow-through (unbound) fraction. By rendering the monitor peptides morenearly equal in abundance (as compared to the very different abundancesthey might have in the sample digest), the dynamic range limitations ofthe LC and the MS cease to be major problems. This combination ofmonitor peptides can then be analyzed directly by introduction to theMS, provided that the masses of the monitor peptides (both natural andlabeled forms) are different enough to allow the MS to resolve andquantitate all monitor peptides (in both forms) individually. Renderinga series of monitor peptides more nearly equimolar is a major advance inallowing multiplex (multianalyte) measurement by mass spectrometry.

Alternatively the combined monitor peptides could be subjected to LC/MSsuch that only one or a few monitor peptides were introduced into the MSat a time, and the MS could be pre-programmed to look for each monitorpeptide in succession. Thus, in order to measure a series of monitorpeptides (representing a series of protein analytes), all thecorresponding isotopically labeled peptide standards are added to thedigest (prepared as above by the computerized fluid handling system),and then the eluted peptides (now consisting of a series of monitorpeptides with their corresponding labeled standards) are introduced intoa chromatography column (such as a C18 reverse phase column, formingpart of a chromatography system also under computer control) and elutedfrom this reverse phase column (typically over 5 to 30 minutes) by agradient (typically of 0-70% acetonitrile in water with 0.05%trifluoroacetatic acid). The output (eluate of the column) is directedinto the mass spectrometer, with the result that only one or a few ofthe monitor peptides appear at any one time, thus allowing the MS tomeasure each individually without the potential for interference of theother monitor peptides. An initial run is performed during which theelution time of each monitor peptide is determined by scanning themasses of all the eluted peptides. In subsequent runs, the LC/MS systemcan be programmed to look at the right elution time for peptides of theknown masses of the labeled and unlabeled version of the monitorpeptides, thereby allowing more measurement time to be directed to thedesired monitor peptide measurements rather than scanning all peptidemasses.

In this way the disclosed method using capture antibodies for enrichmentcan be parallelized to allow measurement of many proteins, and a seriesof proteins of very different relative abundances in the sample can bequantitated in a single MS or LC/MS operation. The equalization of theabundances (“stoichiometry flattening”) of the monitor peptides is thekey concept, and it overcome a key problem with MS quantitation, namelythe limited dynamic range available with current systems.

In a third parallelized embodiment, a series of proteins are to bemeasured that have very different abundances in the sample, but we wishto economize on the use of labeled monitor peptides for the abundantproteins (FIG. 9). For example, serum albumin, complement C2 andthyroglobulin differ in abundance in human serum by steps ofapproximately 1,000-fold (relatively 10⁶:10³:1 in concentration). If asample of plasma is digested to peptides with an enzyme such as trypsin,then the monitor peptides derived from these three proteins will alsodiffer in abundance by the same factors (assuming complete digestion).Now the basis of the quantitative method proposed is to 1) choose atleast one peptide of suitable chemical properties to represent eachprotein to be measured (the monitor peptide); 2) add an isotopicallylabeled version of each monitor peptide at a concentration similar tothat of the natural (sample-derived) peptide, thereby providing aninternal quantitative standard distinguishable by mass in a massspectrometer but otherwise chemically the same as the natural peptide;3) enrich each monitor peptide (natural plus isotope labeled standard)by capture on a specific anti-peptide antibody and elution afterwashing; and 4) analyze the enriched monitor peptides in a massspectrometer to determine the ratio of natural versus isotope labeledpeptide detected. This ratio gives the quantity of the original analyteprotein in relation to the amount of added isotope labeled standard.

In this embodiment, it is noted that since it is optimal to add thelabeled peptide standards (dashed bars, Series 2 in FIG. 9) at aconcentration similar to that of the equivalent natural peptide (solidbars, Series 1 in FIG. 9); i.e., that the internal standard be presentat a similar concentration to the monitor peptide to be quantitated,then the albumin monitor peptide must be added at 1,000 times the amountof the C2 monitor peptide and 1,000,000 times the amount of thethyroglobulin monitor peptide (since these three proteins are typicallypresent at these relative concentrations). Clearly the albumin monitorpeptide, at 1,000,000-fold higher concentration, is over-abundant whenthe thyroglobulin peptide can be detected, and adding 1,000,000 timesthe amount required for the thyroglobulin monitor peptide is wasteful ofthe labeled albumin monitor peptide (which will typically be made bysynthesis). Hence in this embodiment (FIG. 9C), three subsamples of theplasma peptide digest are prepared: one undiluted (to which is add therequired amount of the thyroglobulin labeled monitor peptide, and thoseof other proteins in Group 3 abundance class: FIG. 9), a second dilutedabout 1,000 fold (Group 2, to which we add about the same amount of thelabeled complement C2 monitor peptide), and a third diluted about1,000,000 fold (Group 1, to which is add the albumin monitor peptide).Thus diluted samples are created within which to detect groups ofhigher, middle and low abundance proteins, thereby requiring less of thecorresponding labeled monitor peptides. After capture by the antibodiesand elution, the peptides are recovered at nearly the same relativeabundance (FIG. 9C).

In practice the number of groups (dilutions) will depend on the degreeto which labeled peptides need to be conserved, and may be greater thanthree. In the preferred embodiment this principle is implemented bygrouping the monitor peptides for various abundance classes of proteinsin the sample, into 5 abundance classes, each of which covers only a100-fold range of protein abundance (and which together span10,000,000,000 fold in abundance). The labeled monitor peptides for eachprotein in a class are combined into a cocktail whose members are within100 fold of one another (relative abundances set according to theexpected relative abundances of the proteins in the sample). Anundiluted aliquot and 4 dilutions of the sample peptide digest (each of100-fold relative to the last) are prepared, and the 5 labeled monitorpeptide cocktails are added to the respective dilutions prior to theenrichment and analysis steps (4 and 5 above). The method thus requiresmuch less monitor peptide for high abundance proteins at the cost ofrunning 5 analyses instead of one. For reasons set out below, this iscompatible with other factors to be optimized, and that limit the numberof peptides to be analyzed in one step anyway.

The major element of this embodiment is that peptides are grouped intoclasses according to the expected abundance of the respective targetprotein in the sample. The required abundance information is obtainedfrom exploratory studies using the disclosed invention (scanning aseries of such dilutions to see in which class a given monitor peptidecan be detected) or from other quantitative measurements includingpublished data. The necessary data is organized in a database and usedwith other criteria for the selection of optimal monitor peptide sets,making use of the ability of the database system to filter, rank andsort thousands to millions of candidate in silico peptides by complexcriteria.

In a fourth parallelized embodiment (FIG. 10), the monitor peptides aregrouped into classes based on peptide mass, in such a way that thepeptides in a class do not overlap. An example of such a class criterionis as follows: assume the peptide selected as monitor peptide forprotein a has mass A (in atomic mass units (amu) or daltons) and is forthe sake of this example singly charged (i.e. is detected at an M/Zvalue of A), and that the corresponding isotopically labeled monitorpeptide A has a mass of A+6, and that it can be assumed both peptideshave a series of mass analogs in their spectra extending 5 mass units upfrom the parent masses (due to the well known incorporation of naturalfrequencies of various stable isotopes). The peptide peaks for thesepeptides will thus extend over the range {A, A+1, A+2, A+3, A+4, A+5}for the natural peptide (solid bars in FIG. 10) and {A+6, A+7, A+8, A+9,A+10, A+11} for the stable isotope standard (dashed b 10), or in otherwords over a range of A to A+11 amu. Assume then that one selects amonitor peptide B from another protein b that has a mass of A+12: noneof the mass variant peaks of this peptide B will overlap peaks of themass variants of the peptide A. Similarly one selects a series ofmonitor peptides ideally spaced at least 12 amu apart. Thus a series of50 peptides could be placed in the mass range 1,000 to 1,600. Inpractice the peptides available for selection will not be ideallyarranged and thus it is likely that no more than 10 might be combined tospan the ideal measurement range without overlap. Thus these 10 proteinscould be quantitated at once by the MS, and this class is a panel ofsimultaneously measurable proteins. Since the panel of peptides would beintroduced into the MS at once, an MS that can efficiently scan therequired range of masses at high duty cycle is desired. In the detectionscheme disclosed, these 10 monitor peptides would be added to thenatural sample peptide digest, and the corresponding 10 antibodies wouldbe used together to enrich these 10 peptides for analysis.

The key element of this embodiment is the selection of mutuallycompatible sets of monitor peptides based on non-overlapping masses.

In a fifth parallelized embodiment, the third and fourth above arecombined. Here we select classes of monitor peptides that satisfyboth 1) similarity in parent protein abundance class in the sample, and2) non-overlapping masses. These criteria may be applied in combinationwith the other criteria disclosed in the basic single-analyte embodiment(hydrophilicity, appropriate size, lack of certain amino acids, etc).The classes so developed are optimized for use in the disclosedmeasurement method. They maximize the number of proteins that canquantitated in one MS run and minimize the consumption of the typicallyexpensive stable isotope labeled monitor peptides. The strategies forallowing multiple monitor peptides to be detected at once can becombined with the automation methods to allow large numbers of analytesto be routinely measured.

Other Embodiments

A series of additional embodiments make use of anti-peptide antibodiesin alternative methods.

Another embodiment makes use of experimentally observed partial peptidesequence data obtained as “de novo” sequence by MS/MS techniques formonitor peptide design, instead of sequence derived from an existingdatabase.

Another embodiment (FIG. 11) uses multiple isotopically labeled peptidesfor a given monitor peptide analyte, added at a series of levels tocreate a standard curve. In this embodiment, two or more versions ofeach monitor peptide are synthesized having different isotopic masses.If the peptide sequence contained 3 histidines for example, one versionof the peptide (IV1) might contain a single isotope-labeled HIS residue(6× carbon-13; net 6 dalton heaver than natural), while a second version(IV2) contained two, and a third version (IV3) contained all threehistidines as heavy isotope versions. The three modified peptides wouldthus be 6, 12 and 18 daltons heavier than the natural version. One couldthen add to the sample an amount of IV1 equal to ⅕ the expected amountof the natural version, an amount of IV2 equal to the expected naturallevel, and an amount of IV3 equal to 2 times the natural level. The MSspectrum of the monitor peptide would thus display four separatelyresolved versions, with the three isotopically labeled versionsproviding an internal standard curve covering a 10-fold range around theexpected value. In this way the monitor peptide could be measuredagainst an internal standard curve in every sample. Obviously anycombination of labeled amino acids and appropriate isotope labels couldbe combined to design labeled monitor peptides that differ from thenatural peptide and one another by any of a range of mass increments.

Another embodiment makes use of specific properties engineered into themonitor peptides specifically designed to facilitate labeling withoxygen-18. In this case (assuming that trypsin is used as thefragmenting enzyme) an extended version of the monitor peptide could besynthesized without isotopically labeled amino acids, but in which theadded c-term residues begin with a Lys or Arg residue (e.g.,nterm-MONITOR-lys-gly-ser-gly-cys-cterm as in the first preferredembodiment). By cleaving this peptide with trypsin in the presence ofoxygen-18 water, two atoms of oxygen-18 (4 daltons heavier than natural)would be introduced into the final monitor peptide. The advantage ofthis approach is that only a single version of the monitor peptide wouldneed to be made: the same extended version could be used forimmunization, for affinity purification of the antibody, and as the MSinternal standard (after cleavage and oxygen-18 introduction), therebypotentially reducing cost. Other similar constructs can be constructedappropriate for cleavage by other enzymes if their mechanisms ofcleavage are amenable to the introduction of isotope labels.

Another embodiment makes use of isotopically-labeled monitor peptidesprepared by labeling natural peptides (derived from digestion of areference sample) instead of chemical synthesis (as described in thefirst preferred embodiment). In this approach, a reference sample of thetype to be analyzed subsequently is digested with the same enzyme orreagent as will be used on subsequent samples (e.g., trypsin) to yieldpeptides. These peptides are then reacted with a chemical derivatizingagent containing an isotopic label. For example, the peptides may bereacted with deuterated iodoacetamide, thereby introducing a label onall cysteines-containing peptides. Alternatively the sample proteinscould be reduced and alkylated with deuterated iodoacetamide prior tocleavage by trypsin. Samples to be analyzed would be reduced andalkylated with unlabeled iodoacetamide, so that the sample and labeledmonitor peptides would be chemically the same. The labeled monitorpeptide mixture (essentially the labeled digest of the reference sample)would be added to the digest from a test sample, typically in equalproportions, and the resulting mixture subjected to antibody enrichmentof the selected monitor peptides. In this case the monitor peptideswould be selected from among the cysteine containing peptides of thetarget protein. The resulting MS readout would reveal the ratio inabundance for the monitor peptide (and hence target protein) between thereference sample and the test sample. Since in this embodiment the labelis introduced through a chemical modification of the monitor peptide,the enrichment antibody is preferably raised by immunization with asimilarly modified peptide: the peptide immunogen would be acysteines-containing peptide in which the cys was alkylated withiodoacetamide. Other chemical means can be used to introduce otherlabels on various amino acids, or into the n-terminal or c-terminalgroups specifically. In each case, the same modification would be madeto the test sample peptides (either before or after digestion, asappropriate, but without the isotope label) and the antibody would becreated against the appropriately modified synthetic peptide.

Alternatively a phage-display or other in vitro technique can be used toselect antibodies against the monitor peptide.

A further embodiment makes use of monoclonal antibodies, phage displayantibodies (single chain or Fab), single domain antibodies, affibodies,or other chemically uniform proteins as peptide binding reagents.

In a further alternative, the immunizing peptides are prepared from adigest of the parent protein, rather than by chemical synthesis.

In a further alternative, the immunizing peptides are synthesized asfusion proteins in an expression system (such as E. coli, baculovirus,yeast) or an in vitro translation system (such as rabbit reticulocyte,wheat germ or E. coli lysate), rather than by chemical synthesis. Thedesired peptides can be produced in multiple copies in the fusionprotein if desired, can be isolated through an incorporated affinity tag(e.g., the FLAG peptide, or a polyhistidine tag), and can besubsequently cleaved from the fusion protein (e.g., via trypsin) andpurified by liquid chromatography or other methods. The isotopicallylabeled monitor peptides can be made by similar means through theincorporation of labeled aminoacids into the synthesis system.

In a further alternative, the enriched monitor peptide (natural plusisotope labeled) is applied to a target for MS analysis in a MALDI massspectrometer.

A further embodiment makes use of fluorescence detection instead of MSdetection, and uses covalent fluorescent labels (e.g.,cysteines-reactive Cy3 and Cy5 dye labels) to label the sample peptidesand the added monitor peptides. In this case, the antipeptide antibodiesare created against dye-conjugated antigens and selected in such a waythat they bind the two forms (e.g., Cy3 and Cy5) relatively equally. Thefinal fluorescence ratio detection can be carried out directly uponelution of the peptides from the antibody (if the antibody is specificenough to reject all other peptides), or following another separationstep (e.g., reverse phase LC or capillary electrophoresis) in which theCy3 and Cy5 peptides behave, if not identically, at least in areproducible and decipherable way (so that the dilution positions of thetwo version of the monitor peptide can be confidently predicted formeasurement).

A further embodiment makes use of fluorescence detection in which asynthetic monitor peptide standard is labeled with the label (e.g., Cy5)and the sample digest remains unlabeled. In this embodiment thesample-derived monitor peptide competes with the fluorescently labeledstandard peptide for binding to the corresponding anti-peptide antibody.The concentration of the sample-derived peptide can thus be inferred,using a standard working curve, from the amount of labeled standardpeptide bound to and subsequently eluted from the antibody. At highsample concentrations of parent protein (high concentrations of monitorpeptide in the digest) less labeled standard peptide will be bound, andvice versa. The fluorescently labeled standard peptides can then beseparated and detected very sensitively using capillary electrophoresis,and particularly the multichannel devices developed for DNA sequencing(e.g., ABI 3700, Amersham MegaBACE).

Example

A database of 289 proteins detected in human plasma by various means wasconstructed by combining information from textbooks, catalogs ofdiagnostic assays, and a search of the scientific literature. Amino acidsequences for these proteins were downloaded and stored in a MicrosoftAccess database as text fields. Each protein sequence was processed inan Excel spreadsheet by a macro procedure that created a list of trypticpeptide fragments. A series of parameters was computed for each peptidesequence, including length, mass, expected net charge at neutral pH,total charged groups, HoppWoods hydrophilicity (HWH) and normalized HWH(HWH/number of amino acids), and the numbers of Cys, Trp, Pro and Metresidues. A first selection of usable peptides was made based on thefollowing requirements evaluated by an Excel macro: length>7 and <14residues, no Cys, Met or Trp residues, normalized HWH>−0.5 and <0.5, andmass>800. The results (peptide sequences, and computed parameters) werestored in the database. A total of 10,204 peptides were thus derived, ofwhich 751 met the initial requirements.

For a proof of concept test, one monitor peptide was selected for eachof four protein analytes: TNF, IL-6, hemopexin andalpha-1-antichymotrypsin. Where multiple peptides from a protein met theinitial requirements, preference was given to peptides that contained aproline (as this is expected to increase immunogenicity). Syntheticpeptides were generated for each sequence with a four residue extension(GSGC or CGSG) to allow coupling to keyhole limpet hemocyanine (KLH)carrier via a terminal Cys residue. In the case of the TNF alphapeptide, two versions of the monitor peptide were synthesized: one withan N-term extension and the other with a C-term extension (all otherpeptides carried a C-terminal extension). The peptide sequences(extensions underlined) were:

IL-6 residues 83-94 with C terminal extension GSGC Hemopexin residues92-102 with C terminal extension GSGC Alpha-1-Antichymo residues 307-314with C terminal extension GSGC TNF-a C-term residues 66-78 with Cterminal extension GSGC

Each peptide was coupled to an albumin carrier and injected into tworabbits according to a short immunization schedule. Antibody productionis monitored via an ELISA assay using peptide immobilized on microwellplates. For each peptide, polyclonal antibody from the better of the tworabbit antisera (based on higher ELISA signal indicating more or higheraffinity antibody) was immunoaffinity purified on a column of properlyoriented peptide immobilized on thiol containing Sepharose.

Immunopurified antibodies were immobilized in an oriented manner onPOROS protein G resin (Applied Biosystems). Once the antibody associatedby specific interaction of protein A with the Fc portion of the antibodymolecules, covalent crosslinking was achieved by exposure todimethylpimerimidate (DMP) according to the manufacturer's instructions.The anti-peptide antibody POROS (APA-POROS) support was washed andstored at 4 C.

Capillary microcolumns (1 cm×100 microns) containing the supports arepacked in pre-made frit-fitted capillaries (New Objectives) using a bombpressurized with 1000 psig He. Supports carrying rabbit polyclonal AB tothe first four peptides tested above were individually exposed to amixture of the four respective labeled monitor peptides. The supportswere washed and the bound peptides eluted using 10 microliters of 10%acetic acid to a capillary C18 referce phase column and thereaftereluted ionto the ESI source of a Qtrap (applied Biosystems) MS system bya 0% to 70% ACN gradient in 0.05 formic acid. On average, the antibodysupports showed a 100-fold enrichment of the ‘correct’ monitor peptide.

1. A method of quantifying an amount of at least a first monitor peptideand a second monitor peptide in a biological sample, comprising:contacting the sample with (i) a first anti-peptide antibody specificfor said first peptide and; (ii) a known quantity of a labeled versionof said first peptide; contacting the sample with (i) a secondantipeptide antibody specific for said second peptide, wherein saidsecond antibody is different from said first antibody and; (ii) a knownquantity of a labeled version of said second peptide, separatingpeptides bound by said first and said second antibodies from unboundpeptides; eluting said peptides bound by said first and said secondantibodies from said antibodies; measuring the amount of said firstpeptide eluted from said first antibody using a mass spectrometer;measuring the amount of said labeled version of said first peptideeluted from said first antibody using a mass spectrometer; calculatingthe amount of the first peptide in the biological sample; measuring theamount of said second peptide eluted from said second antibody using amass spectrometer; measuring the amount of the labeled version of thesecond peptide eluted from said second antibody using a massspectrometer; and calculating the amount of the second peptide in thebiological sample, wherein said biological sample is a proteolyticdigest of a bodily fluid sample.
 2. The method of claim 1, wherein atleast one of said first and said second antibodies is a monoclonalantibody.
 3. The method of claim 1, wherein at least one of said firstand said second antibodies is a polyclonal antibody.
 4. The method ofclaim 1, wherein said first and said second antibodies are bothpolyclonal antibodies.
 5. The method of claim 1, wherein said first andsaid second antibodies are both monoclonal antibodies.
 6. The method ofclaim 1, wherein the labeled version of the first peptide includes atleast one site at which a stable isotope is substituted for thecorresponding predominant natural isotope in more than 98% of peptidemolecules.
 7. The method of claim 1, further comprising: attaching thefirst antibody to a support.
 8. The method of claim 1, furthercomprising: attaching the first antibody to a packed column.
 9. Themethod of claim 1, further comprising: attaching the first antibody to amonolithic porous support.
 10. The method of claim 1, furthercomprising: attaching the first antibody to a mesh.
 11. The method ofclaim 1, further comprising: attaching the first antibody to magneticbeads.
 12. The method of claim 1, wherein the first peptide and thesecond peptide are selected from among the set of peptides produced bydigestion of the target protein to provide high signal to noise in themass spectrometer.
 13. The method of claim 1, further comprising:preparing the labeled version of the monitor peptide.
 14. The method ofclaim 13, wherein the labeled version of the monitor peptide includes astable isotope.
 15. The method of claim 1, wherein said firstanti-peptide antibody is created using said first peptide or anonmaterially modified version of the first monitor peptide.
 16. Themethod of claim 1, further comprising: creating the first antibody usingthe first peptide or a non-materially modified version of the firstpeptide.
 17. The method of claim 1, wherein the said bound peptides aresubjected to a chromatography step after elution from said antibodiesand before introduction into said mass spectrometer.
 18. The method ofclaim 1 wherein said first and second peptides are proteolyticallycleaved from first and second sample proteins, respectively, and whereinthe amounts of said first and second proteins in said body fluid sampleare calculated from the amounts of said first and said second peptidesin the sample.
 19. The method of claim 1, wherein said flit monitorpeptide is a peptide fragment of TNF or IL-6.
 20. A method forquantifying the amount of a peptide in a biological sample, comprising:contacting the sample with (i) an anti-peptide antibody specific forsaid peptide; (ii) a known quantity of a labeled version of the peptide,separating peptides bound by said antibody from unbound peptides elutingsaid peptide bound by said antibody from said antibody; measuring theamount of the peptide eluted from said antibody using a massspectrometer: and calculating the amount of the peptide in thebiological sample; wherein said biological sample is a proteolyticdigest of a bodily fluid.
 21. The method of claim 20, furthercomprising: preparing the labeled version of the peptide.
 22. The methodof claim 20, wherein the labeled version of the peptide includes atleast one site at which a stable isotope is substituted for thepredominant natural isotope in more than 98% of peptide molecules. 23.The method of claim 20, further comprising: creating the anti-peptideantibody using the peptide or a non-materially modified version of thepeptide.
 24. The method of claim 20, wherein said bound peptides aresubjected to a chromatography step after elution from said antibody andbefore introduction into said mass spectrometer.
 25. The method of claim20, wherein the anti-peptide antibody is a polyclonal antibody.
 26. Themethod of claim 20, wherein the anti-peptide antibody is a monoclonalantibody.
 27. The method of claim 20 wherein said first and secondpeptides are proteolytically cleaved from first and second sampleproteins, respectively, and wherein the amounts of said first and secondproteins in said body fluid sample are calculated from the mounts ofsaid first and said second peptides in the sample.
 28. The method ofclaim 20, wherein the polyclonal antibody is created using the monitorpeptide or a non-materially modified version of the monitor peptide.